Glioblastoma (GBM) is a uniformly fatal brain cancer with poor response to treatment and virtually inevitable recurrence. GBM tumors do not metastasize. Instead, they aggressively infiltrate the brain. Tumor borders are highly vascularized by microvessels that serve as key niche environments promoting GBM cell survival, treatment resistance and migration. Currently, it is believed that a subpopulation of treatment-resistant, stem- like GBM cells (GSCs) migrate away from primary tumors ? remaining in close contact with local microvessels ? and seed recurrent tumors ? which are enriched in GSCs. However, the mechanisms underlying the influence of the microvessel niche on GBM cell migration away from primary tumors, leading to recurrence, remain largely unknown. To address this gap in knowledge, the proposed studies will investigate 1) the role of specific cues in the tumor microenvironment on the migratory phenotype of GBM cells and 2) how these cues differentially affect distinct subpopulations of cells within heterogeneous tumors, including GSCs. Our fundamental hypothesis is that the microenvironment surrounding tumor microvessels promotes GBM progression through both mechanical and chemical means. Aim 1 will investigate effects of mechanical cues and Aim 2 effects of biochemical cues present in the microvessel niche on migratory phenotype of GBM. To do this, we will use an innovative, tissue- engineered platform for 3D culture of patient-derived GBM cells to quantify how specific features of the microenvironment affect migratory behavior. We propose to modularly tune mechanical and chemical properties of culture platforms to characterize their independent effects on migratory phenotype of GBM. For some experiments, platforms will be constructed with an interface between defined microenvironments through which GBM cells can migrate, enabling separation of migratory and non-migratory cells for downstream analysis and providing information about how subpopulations of cells within a single tumor, such as GSCs, may respond differentially to microenvironmental cues. As treatment resistance, invasion and recurrence are inter-related events, we will investigate how exposure to routine clinical therapies may alter migratory responses of GBM to microenvironmental cues. Using single-cell RNA sequencing, we will investigate the mechanisms underlying responses to specific features of the microvessel niche. Collectively, the proposed studies will make significant strides towards our understanding of how interactions of GBM with the microvessel niche promote tumor recurrence by characterizing: 1) biomechanical properties of GBM tumors and microvessels (Aim 1), 2) migratory response of GBM cells to biochemical and biomechanical cues (Aims 1 and 2) and 3) mechanisms governing the migratory responses to biochemical and biomechanical cues (Aims 1 and 2). In the long term, we expect these insights will lead to the development of new therapies targeting GBM recurrence. !